脓毒症对大鼠骨骼肌组织泛素及泛素化蛋白表达的影响

Effect of sepsis on the expression of ubiquitin and ubiquitinated protein in rat skeletal muscle

目的 研究脓毒症大鼠骨骼肌组织泛素及泛素化蛋白表达的变化规律 ,探讨导致骨骼肌蛋白降解增强的机理。 方法 大鼠腹腔注射内毒素复制脓毒症模型 ,将其随机分成内毒素攻击后 2、6、12和 2 4h组 ,每组 (16只 )各设正常对照。骨骼肌充分氧供离体孵育后 ,氨基酸自动分析仪测定脓毒症大鼠伸趾长肌总蛋白降解率和肌纤维蛋白降解率 ,利用蛋白免疫印记 (westernblot)方法测定大鼠不同时相点伸趾长肌泛素及泛素化蛋白的表达变化。 结果 脓毒症大鼠伸趾长肌总蛋白降解率在内毒素攻击后 2h和 6h轻度增加 ,12h和 2 4h增加不明显 ,而肌纤维蛋白降解率则在 2h和 6h分别增加 15 5 %和 2 2 2 % ,12h增加 40 % ,2 4h变化不明显 ;伸趾长肌泛素及泛素化蛋白的表达在内毒素攻击后 2h和 6h表达较正常对照组明显增加 ,其中游离泛素蛋白增加 46 % ,泛素化蛋白增加 2 4倍 ,但 12h和 2 4h无明显变化 ,增加的泛素化蛋白主要为高分子量蛋白。 结论 脓毒症大鼠骨骼肌泛素 蛋白酶体途径被激活 ,泛素及泛素化蛋白高水平表达表明进入泛素 蛋白酶体途径底物显著增多 。

Objective To study the regularity of ubiquitin and ubiquitinated protein expression in rat skeletal muscle during sepsis, and the molecular mechanism of enchancement in skeletal muscle protein catabolism. Methods Wistar rats with sepsis were administered endotoxin peritoneally. The rats were randomly divided into 4 groups rats: 2, 6, 12 and 24 h after administration of endotoxin; each group (16) included normal controls. In vitro muscle incubation system with sufficient oxygen supply was used with amino acid automatic analyzer for detecting the proteolytic rate of the extensor digitorium longus(EDL) and soleus(SOL) muscle in the sample. The expression of ubiquitin and ubiquitinated protein in rat EDL muscle was determined by western blot. Results Total proteolytic rate in the EDL muscle increased slightly at 2 and 6 h after administering endotoxin into the peritoneal cavity, and no significant difference at 12 and 24 h was observed. There was a progressive increase of 155% and 220% in myofibrillar proteolytic rate at 2 and 6 h, and 40% at 12 h, respectively, as compared with that of the normal controls. The expression of ubiquitin and ubiquitinated protein in the EDL muscle rose by 46% and 2 4 fold at 2 h and 6 h after administering endotoxin, while the ubiquitinated protein of high molecular weight was determined. No significant changes were noted in the expression of ubiquitin and ubiquitinated protein at 12 and 24 h. Conclusion The results indicate that activation of ubiquitin proteasome pathway occurs in rat skeletal muscle during sepsis, and high expression of ubiquitin and ubiquitinated protein means that the substrate flowing into ubiquitin proteasome pathway increases markedly, and then leads to muscle wasting.